B-001 Method Development of C-Reactive Protein in the Serum of Non-Human Primates

Abstract

Abstract Objective The objective of this study is to validate C-Reactive Protein (CRP) measurement in Non-Human Primate (NHP) serum using the ADVIA 1800 analyzer. Methods CRP was validated in NHP serum using the ADVIA 1800 analyzer. Validation testing included intra-assay precision, inter-assay precision, accuracy, linearity of dilution, limit of quantitation (LOQ), limit of detection (LOD), reference interval, carry-over, correlation, and stability. Samples were collected into tubes containing no anti-coagulant. Intra-assay precision was determined by analyzing two biological samples and the two quality control levels of Bio-Rad Lyphochek Immunology Plus Control 10 consecutive times (in duplicate) within a single run. Inter-assay precision was determined by analyzing two biological samples and the two quality control levels of Bio-Rad Lyphochek Immunology Plus Control six times for six days over a 10-day period. Accuracy was calculated using the data obtained from the inter-assay precision testing. For linearity of dilution, calibrator material and diluted specimen samples were analyzed in duplicate. The LOQ was determined by diluting the low-level calibrator material to produce a value at the low end of the reportable range. Diluted material was tested six times within the same run. The LOD was determined by assaying the appropriate blank 10 times within the same run. The reference interval was determined by analyzing 21 serum samples. Correlation between the two ADVIA 1800 analyzers was determined by assaying 10 samples on both analyzers once. The carry-over of the assay was determined by analyzing the high-level quality control material followed by the low-level quality control material. Stability of serum samples was tested at room temperature, refrigerated, and frozen (at -20°C) for various durations of storage. Results Results show that measurement of CRP in NHP serum met the acceptance criteria for intra-assay precision, inter-assay precision, accuracy, linearity of dilution, limit of quantitation, carry-over, and stability. For intra-assay precision in specimen and control material, the %CV was within ±20%. The inter-assay precision for specimen and control material %CV was within ±20%. The total error observed (TEobs) was within ±20% for accuracy in control material and the obtained mean was within the acceptable range specified by the manufacturer. For linearity of dilution, the coefficient of determination was ≥0.9000 and TEobs for each dilution and calibrator level was lower than 20%. The LOQ was set at the lowest concentration for which the TEobs was within ±20%. The LOD was calculated by adding the mean concentration obtained and three times the standard deviation. The reference interval was set to the 2.5th to 97.5th percentile interval using Excel software. The carry-over was acceptable as it was ≤2%. Correlation between the two ADVIA 1800 analyzers was determined to describe any bias in result interpretation. Stability at all storage conditions was found to be acceptable with a mean %difference within ±20%. Conclusion CRP in NHP serum met the acceptance criteria for all required validation parameters. The ADVIA 1800 analyzer can be used for preclinical studies to test CRP in NHP serum.